Early post-transfection isolation of cells (epic) for biologics production

ABSTRACT

Provided herein are methods for selecting a population of cells expressing a target polypeptide. In some aspects, the disclosure provides methods for sorting and selecting populations of transfected host cells based on their early expression of a selectable polypeptide. In certain embodiments, the sorting is performed using fluorescence-activated cell sorting or magnetic-activated cell sorting based on the selectable polypeptide. Such selection methods can be further utilized to generate clonal populations of producer cells, e.g. for large-scale manufacturing of a target polypeptide of interest.

RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.16/683,837, filed Nov. 14, 2019, which is a continuation of U.S. patentapplication Ser. No. 15/727,272, filed Oct. 6, 2017, which claims thebenefit of U.S. Provisional Patent Application Ser. No. 62/405,392,filed Oct. 7, 2016, the entire contents of which are incorporated hereinby reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in XML format and is hereby incorporated byreference in its entirety. Said XML file, created on May 10, 2023, isnamed 743098_SA9-179CCON2_ST26.xml and is 17,750 bytes in size.

BACKGROUND

Methods for selection of producer cell populations and cell clones areimperative for the manufacturing of biologics, such as antibodies andfusion proteins. Such methods generally rely on use of a selectionagent, such as methotrexate (MTX) or methionine sulphoximine (MSX), tobias and amplify the production of biologics. Selection agent-basedmethods may affect the viability or growth rate of selected populationsor may have a negative impact on clonal stability. Such drug-basedselections can also be time consuming, often requiring multiple roundsof selection to obtain populations which contain clones that aresuitable for biologic manufacturing. There remains a need for rapid andreliable methods of generating both large cell populations and clonesthat produce high titers of biologics with less negative impact to thehost cell.

SUMMARY OF THE INVENTION

In some aspects, the disclosure provides methods of selecting apopulation of cells expressing a target polypeptide. As describedherein, methods for selection were developed that relied upon sorting ofpopulations shortly following their transfection. Thus the methodsfeature the step of isolating a sub-population of transfected cells forearly detectable expression of the transfected vector. In certainembodiments, the selection is based on early expression of a selectablepolypeptide, which is different from the target polypeptide anddetectable on the surface of the cell.

Unexpectedly, the methods described herein were found to be faster thantraditional methods which use two rounds of MTX selection to generate apool, and more productive than traditional MTX amplification, includingsingle-round MTX selection.

The methods described herein are useful, e.g., for the generation ofpools of cells for screening of polypeptides of interest (such as inearly clinical development and for the generation of high titer clones,which can be utilized to produce a polypeptide of interest both forsmall and large scale manufacturing.

Accordingly, in some aspects, the disclosure provides a method ofproducing a population of producer cells expressing a targetpolypeptide, the method comprising: (a) transfecting host cells with oneor more vectors that encode one or more mRNAs, wherein the one or moremRNAs encode a selectable polypeptide and the target polypeptide; (b)isolating from the transfected host cells, within 2 to 15 days aftertransfection, a sub-population of early-expressing transfected hostcells which express the selectable polypeptide; and (c) expanding thesub-population of early-expressing transfected host cells, therebyproducing a population of producer cells.

In some embodiments, step (b) is performed in drug-selection-freemedium.

In some embodiments, step (c) is performed in drug-selection-freemedium.

In some embodiments, step (b) and step (c) are each performed indrug-selection-free medium.

In some embodiments of any one of the methods provided, the methodfurther comprises isolating the target polypeptide from the expandedsub-population.

In some embodiments of any one of the methods provided, the methodfurther comprises isolating one or more single transfected host cellsfrom the expanded sub-population and culturing the one or more singletransfected host cells to produce clonal populations of the one or moresingle transfected host cells.

In some embodiments of any one of the methods provided, at least one ofthe clonal populations of the one or more single transfected host cellsyields a 2- to 30-fold improvement in production of the targetpolypeptide compared to that of a stable pool of transfected butuncloned host cells obtained at step (c).

In some embodiments of any one of the methods provided, the transfectedhost cells subject to isolation in step (b) contains 80-120×10⁶ cells.

In some embodiments of any one of the methods provided, the isolation instep (b) is performed less than six days after transfection. In someembodiments of any one of the methods provided, the isolation in step(b) is performed between two and four days after transfection. In someembodiments of any one of the methods provided, the isolation in step(b) is performed two days after transfection. In some embodiments of anyone of the methods provided, the isolation in step (b) is performedthree days after transfection.

In some embodiments of any one of the methods provided, thesub-population of transfected host cells contains 0.5-6.0×10⁶ cellsprior to expansion in step (c).

In some embodiments of any one of the methods provided, the expanding instep (c) is for between 4-31 days.

In some embodiments of any one of the methods provided, a first of theone or more vectors encodes the mRNA encoding the target polypeptide,and a second of the one or more vectors encodes the selectablepolypeptide.

In some embodiments of any one of the methods provided, the mRNAencoding the target polypeptide and the mRNA encoding the selectablepolypeptide are both encoded on one vector.

In some embodiments of any one of the methods provided, a first of theone or more vectors encodes the mRNA encoding the target polypeptide,and a second of the one or more vectors encodes the selectablepolypeptide.

In some embodiments of any one of the methods provided, the mRNAencoding the plurality of target polypeptides and the mRNA encoding theplurality of selectable polypeptides are both encoded on one vector.

In some embodiments of any one of the methods provided, the isolation instep (b) employs magnetic-activated cell sorting (MACS),fluorescence-activated cell sorting (FACS), or ClonePix.

In some embodiments of any one of the methods provided, the selectablepolypeptide is a FACS selectable polypeptide and the isolation in step(b) employs FACS.

In some embodiments of any one of the methods provided, the targetpolypeptide and the selectable polypeptide form a fusion polypeptide.

In some embodiments of any one of the methods provided, the mRNA is amulticistronic mRNA. In some embodiments of any one of the methodsprovided, the multicistronic mRNA comprises a first open reading frame(ORF) that encodes the selectable polypeptide and a second ORF thatencodes the target polypeptide, wherein the first ORF is 5′ to thesecond ORF. In some embodiments of any one of the methods provided, thefirst ORF has a non-AUG start codon. In some embodiments of any one ofthe methods provided, the second ORF has an AUG start codon. In someembodiments of any one of the methods provided, the non-AUG start codonis a UUG, GUG, or CUG in a Kozak consensus sequence. In some embodimentsof any one of the methods provided, the ORF that encodes the selectablepolypeptide is devoid of any AUG sequences.

In some embodiments of any one of the methods provided, the selectablepolypeptide is CD52 or CD59.

In some embodiments of any one of the methods provided, the targetpolypeptide is a therapeutic agent. In some embodiments of any one ofthe methods provided, the target polypeptide is a secreted protein. Insome embodiments of any one of the methods provided, the targetpolypeptide is an antibody or an Fc fusion protein.

In some embodiments of any one of the methods provided, the host cellsare CHO cells, HEK293 cells, or HeLa cells.

Other aspects of the disclosure relate to a clonal population oftransfected host cells that express a selectable polypeptide and atarget polypeptide obtainable by any one of the methods described aboveor otherwise described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic depicting comparison between traditionaltransfection and selection and EPIC-based transfection and selection.Early expression refers to expression early after transfection, prior tosignificant genomic integration.

FIG. 1B is a diagram showing reporter expression of a transfectedpopulation of cells from day 3 to 21 in a nucleotide-deficient selectionprocess (compared to mock transfected population). Transfected cellsexhibited an apparent early expression shortly after transfection (e.g.,day 3-4) and then transitioned to stable expression upon completion ofselection (day 18-21).

FIG. 2 is a series of FACS histogram offsets depicting the earlyexpression of both red fluorescent protein (RFP) and cell surfacereporter CD52 expression from the same vector (pGZ729-RFP). No selectionpressure was applied to the transfected cells. Peak early expression forRFP and CD52 occurs between days 2 and 3.

FIG. 3 is a series of FACS histogram offsets depicting the day 3 earlyexpression of RFP and CD52 in cells transfected with pGZ729-RFP(encoding both selectable polypeptide CD52 and target polypeptide RFP)or pGZ700-RFP (encoding only target polypeptide RFP).

FIG. 4 is a schematic showing both the methodology of EPIC to generate asub-population of cells for selection shortly after transfection and thebeneficial effects to both the reporter expression and monoclonalantibody (mAb) titers upon isolation/expansion of the sort-enrichedpopulation. Mock refers to mock transfection.

FIG. 5 is a graph depicting day 14 unfed batch titers for EPIC-generatedpools as compared to traditional MTX methodologies. Pools generated bythe rapid bulking process are also shown (RB #1 and RB #2).

FIG. 6 is a graph depicting day 14 unfed batch titers fromEPIC-generated clones which achieved top expression ranging from 1.5-2.0g/L. Leftmost bar (0.5 g/L) represents titer for EPIC-sorted pool priorto cloning. All other vertical bars represent titers for individualclones.

FIG. 7 is a series of histogram offsets depicting the comparativebenefit of EPIC targeting to generate stable pools transfected withpGZ729-RFP. EPIC was used to target early RFP expression at day 2 whichyielded a stable pool with improved RFP (and CD52 reporter expression)as compared to traditional transfection/selection methodologies (0 nMMTX).

FIG. 8 is a schematic showing the 3 different methodologies forgenerating pools to support a clone limiting dilution (CLD) process. Allmethodologies use similar “pooled” recovering transfected cells to beginthe process. EPIC and Rapid Bulking, which are both MTX-independentprocesses, also have reduced timelines compared to the traditional MTXselection process. This entire scheme was completed for each of threerecombinant proteins (mAb #1, mAb #2, FcFusion #1).

FIG. 9 is a graph showing clonal productivity from three differentmolecules using from pools generated from each process shown in FIG. 8(EPIC, Rapid Bulking, and MTX). The graph illustrates that bothMTX-independent processes (EPIC and Rapid Bulking) achieved clones ofsimilar high productivity to those produced from the MTX selectionprocess.

FIG. 10 is a graph illustrating the timelines of each process from DNAto Pools and Pools to Clones for three different molecules representingeach process (EPIC, Rapid Bulking, and MTX). EPIC pool generations canbe achieved much faster (1 month) than MTX generated pools whichtranslates to significantly shorter overall timelines for production ofclones.

FIG. 11A is FACS histogram overlay showing the variable sort targets ofthe transiently positive population which was isolated to generate anEPIC pool (compared to an unsorted control). FIG. 11B is a graph showingthe productivity associated with each EPIC sort target and clearlydemonstrates that higher transient targeting yields greaterproductivity. The data supports the assertion that EPIC pools enrichmentis solely the result of isolating transiently positive populations.PoP=Proof of Principle

DETAILED DESCRIPTION

Before the present invention is described, it is to be understood thatthis invention is not limited to particular methods and experimentalconditions disclosed herein; as such methods and conditions may vary. Itis also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly by the appended claims.

Furthermore, the practice of the invention employs, unless otherwiseindicated, conventional molecular and cellular biological andimmunological techniques within the skill of the art. Such techniquesare well known to the skilled worker, and are explained fully in theliterature. See, e.g., Ausubel, et al., ed., Current Protocols inMolecular Biology, John Wiley & Sons, Inc., NY, N.Y. (1987-2008),including all supplements; M. R. Green and J. Sambrook, MolecularCloning: A Laboratory Manual (Fourth Edition), Cold Spring HarborLaboratory, Cold Spring Harbor, New York (2012); and Harlow et al.,Antibodies: A Laboratory Manual, Chapter 14, Cold Spring HarborLaboratory, Cold Spring Harbor, New York (2013, 2n d edition).

I. Definitions

Unless otherwise defined herein, scientific and technical terms usedherein have the meanings that are commonly understood by those ofordinary skill in the art. In the event of any latent ambiguity,definitions provided herein take precedent over any dictionary orextrinsic definition. Unless otherwise required by context, singularterms shall include pluralities and plural terms shall include thesingular. The use of “or” means “and/or” unless stated otherwise. Theuse of the term “including”, as well as other forms, such as “includes”and “included”, is not limiting.

Generally, nomenclatures used in connection with cell and tissueculture, molecular biology, immunology, microbiology, genetics andprotein and nucleic acid chemistry and hybridization described hereinare those well-known and commonly used in the art. The methods andtechniques provided herein are generally performed according toconventional methods well known in the art and as described in variousgeneral and more specific references that are cited and discussedthroughout the present specification unless otherwise indicated.Enzymatic reactions and purification techniques are performed accordingto manufacturer's specifications, as commonly accomplished in the art oras described herein. The nomenclatures used in connection with, and thelaboratory procedures and techniques of, analytical chemistry, syntheticorganic chemistry, and medicinal and pharmaceutical chemistry describedherein are those well-known and commonly used in the art. Standardtechniques are used for chemical syntheses, chemical analyses,pharmaceutical preparation, formulation, and delivery, and treatment ofpatients.

That the disclosure may be more readily understood, select terms aredefined below.

As used herein, the term “polynucleotide” intends a polymeric form ofnucleotides of any length, examples of which include, but are notlimited to, a gene or gene fragment, exons, introns, messenger RNA(mRNA), transfer RNA, ribosomal RNA, ribozymes, complementary DNA(cDNA), recombinant polynucleotides, branched polynucleotides, plasmids,vectors, isolated DNA of any sequence, isolated RNA of any sequence,nucleic acid probes, and primers. A polynucleotide may comprise modifiednucleotides, such as methylated nucleotides and nucleotide analogs.

As used herein, the term “polypeptide” intends a polymeric form of aminoacids of any length, examples of which include, but are not limited to,a protein, a protein fragment, a multimeric protein, a fusion protein,an antibody (including fragments thereof), and a peptide.

As used herein, a “selectable polypeptide” is a polypeptide that can bedetected, directly or indirectly, by any suitable method including, forexample and without limitation, fluorescence-activated cell sorting(FACS), magnetic-activated cell sorting (MACS), ClonePix, and affinitychromatography. In certain embodiments, the selectable polypeptide isexpressed on the surface of a cell, i.e., is a cell surface polypeptide.Examples of selectable polypeptides include polypeptides that include anextracellular domain (e.g., CD52 or CD59) that are capable of beingbound to or by a detectable binding partner (e.g., afluorescently-labeled antibody). Other examples of selectablepolypeptides include fluorescent proteins such as green fluorescentprotein (GFP), red fluorescent protein (RFP), yellow fluorescent protein(YFP), blue fluorescent protein (BFP), and variants thereof includingeGFP, Venus, mCherry, mTomato, and the like. In certain embodiments, theselectable polypeptide may be conveniently detected, directly orindirectly, by flow cytometry.

As used herein, “fluorescence-activated cell sorting” or “FACS” refersto a method of separating a population of cells into one or moresub-populations based on the presence, absence, or level of one or moreFACS-selectable polypeptides expressed by the cells. FACS relies onoptical properties, including fluorescence, of individual cells in orderto sort the cells into sub-populations. FACS cell sorters suitable forcarrying out a method described herein are well-known in the art andcommercially available. Exemplary FACS cell sorters include BD Influx™(BD Biosciences) and other equivalent cell sorters produced by othercommercial vendors such as Sony, Bio-Rad, and Beckman Coulter.

As used herein, a “FACS selectable polypeptide” is a polypeptide thatcan be detected, directly or indirectly, by flow cytometry. Examples ofFACS selectable polypeptides include polypeptides that include anextracellular domain (e.g., CD52 or CD59) that are capable of beingbound to a detectable binding partner (e.g., a fluorescently-labeledantibody) for indirect detection of the polypeptide by flow cytometry.Other examples of FACS selectable polypeptides include fluorescentproteins such as green fluorescent protein (GFP), red fluorescentprotein (RFP), yellow fluorescent protein (YFP), blue fluorescentprotein (BFP), and variants thereof including eGFP, Venus, mCherry,mTomato, and the like, which may be detected directly by flow cytometry.

As used herein, magnetic-activated cell sorting, or “MACS” refers to amethod of separating a population of cells into one or moresub-populations based on the presence, absence, or level of one or moreMACS-selectable polypeptides expressed by the cells. MACS relies onmagnetic susceptibility properties of tagged individual cells in orderto sort the cells into sub-populations. MACS cell sorters suitable forcarrying out a method described herein are well-known in the art andcommercially available. Exemplary MACS cell sorters include MACSQuant®flow cytometer (Miltenyi Biotec).

As used herein, a “MACS selectable polypeptide” is a polypeptide thatcan be detected, directly or indirectly, by magnetic-activated cellsorting. Examples of MACS selectable polypeptides include polypeptidesthat include an extracellular domain (e.g., CD52 or CD59) that arecapable of being bound to a magnetically susceptible binding partner(e.g., an iron-, nickel-, or cobalt-labeled bead coupled to an antibody)for direct or indirect detection of the polypeptide. In certainembodiments, the selectable polypeptide may be conveniently detected,directly or indirectly, by flow cytometry.

As used herein, “ClonePix” refers to a method of, and device for,separating a population of cells into one or more sub-populations basedon the presence, absence, or level of one or more selectablepolypeptides expressed by the cells. ClonePix relies on opticalproperties, including white light and fluorescence detection, ofindividual cells or colonies of cells in order to sort the cells intosub-populations. ClonePix is described in U.S. Pat. Nos. 7,776,584;8,034,612; 8,034,625; 8,293,520; 8,293,525; 8,293,526; and 8,293,527,each to Richmond et al., and is commercially available from MolecularDevices (Sunnyvale, CA).

As used herein, “target polypeptide” refers to a protein, a proteinfragment, a multimeric protein, a fusion protein, an antibody (includingfragments thereof), or a peptide that can be produced in host cells andin the aspects exemplified herein, the target polypeptide is selectedbecause of its potential as a therapeutic agent, e.g., an antibody(including a fragment thereof), a Fc fusion protein, a hormone or anenzyme. In some embodiments, the target polypeptide is a secretedprotein. However, the methods described herein are not limited for theselection and scale-up of therapeutic polypeptides. For example,diagnostic polypeptides or polypeptides for use in the environment arealso contemplated for use as a target polypeptide in a method disclosedherein.

In certain embodiments, the selectable polypeptide is a cell surfacepolypeptide, and the target polypeptide is a secreted polypeptide.

As used herein, the term “antibody” refers to such assemblies (e.g.,intact antibody molecules, antibody fragments, or variants thereof)which have significant known specific immunoreactive activity to anantigen of interest. Antibodies and immunoglobulins comprise light andheavy chains, with or without an interchain covalent linkage betweenthem. Basic immunoglobulin structures in vertebrate systems arerelatively well understood.

As used herein, the term “antibody” includes entire antibodies as wellas antigen-binding fragments and variants of such antibodies. Antibodiesmay be of any class, such as IgG, IgA or IgM; and of any subclass, suchas IgG1 or IgG4. The antibody can be a polyclonal or a monoclonalantibody, or it can be fragments of the polyclonal or monoclonalantibody. The antibody can be chimeric, humanized, totally human,bi-specific, or bi-functional. Any antigen-binding fragment or variantof an antibody is also contemplated, such as Fab, Fab′, F(ab′)₂,single-chain variable regions (scFv) and variations of the same.

As used herein, an “Fc fusion protein” refers to a protein comprising animmunoglobulin Fc domain that is linked, directly or indirectly, to apolypeptide, such as a protein or peptide. The linked polypeptide can beany proteinaceous molecule of interest, such as a ligand, a receptor, oran antigenic peptide.

As used herein, the term “producer cell” refers to a cell expressing apolypeptide of interest. In certain embodiments, a producer cell is acell expressing a target polypeptide as disclosed herein. In certainembodiments, a producer cell is a cell expressing both a selectablepolypeptide and a target polypeptide as disclosed herein.

In certain embodiments, the term “producer cells” refers to cells thatare suitable for production of proteins, e.g., in a small- orlarge-scale manufacturing method for producing biologics. In someembodiments, producer cells are mammalian or insect cells. Producercells are further discussed herein.

As used herein, a “population of producer cells” is a population ofcells that expresses an enhanced level of one or more polypeptides,e.g., a FACS selectable polypeptide and a target polypeptide that areencoded by the same multicistronic mRNA. In certain embodiments, a“population of producer cells” is a population of cells that expressesan enhanced level of a target polypeptide. In some embodiments, theenhanced level is at least 10-fold, at least 100-fold, at least1,000-fold, or at least 10,000-fold of the one or more polypeptides inan unselected population. In some embodiments, the enhanced level is atleast 10-fold, at least 100-fold, at least 1,000-fold, or at least10,000-fold of a FACS-selectable polypeptide in an unselected populationas detected by flow cytometry (e.g., on a BD Influx™ cell sorter). Insome embodiments, the enhanced level is at least 10-fold, at least100-fold, at least 1,000-fold, or at least 10,000-fold of aMACS-selectable polypeptide in an unselected population as detected byflow cytometry (e.g., on a MACSQuant® flow cytometer (Miltenyi Biotec)).Methods for generating populations of producer cells are describedherein.

As used herein, a “population of producer cells” is a population ofcells that expresses detectable levels of one or more polypeptides,e.g., a FACS selectable polypeptide and a target polypeptide that areencoded by the same multicistronic mRNA. Methods for generatingpopulations of producer cells are described herein.

As used herein, a “multicistronic mRNA” is an mRNA that contains atleast two open reading frames (ORFs) that are capable of encoding two ormore polypeptides.

As used herein, a “drug-selection-free medium” is a culture medium thatis devoid of a drug (e.g., methotrexate (MTX)) that is used to select apopulation or sub-populations of cells that express a protein thatconfers drug resistance (e.g., dihydrofolate reductase) to thepopulation or sub-population.

As used herein, “medium-based selection” is a selection process by whichthe culture medium is altered to include a selection agent (e.g., MTX)or to exclude a component of medium, which results in selection of asub-population that is resistant to the selection agent or can survivein the absence of the excluded medium component.

As used herein, “nucleotide-deficient medium” is culture medium that isdevoid of or contains low levels (e.g., less than 10 micrograms/mL) ofnucleotides having one or more of the nucleobases adenine (A), cytosine(C), guanine (G), thymine (T), hypoxanthine, or thymidine. In someembodiments, nucleotide-deficient medium is medium that is devoid ofhypoxanthine and thymidine. Exemplary nucleotide-deficient mediumincludes CD CHO Medium (Gibco, Life Technologies, Catalogue numbers10743 (liquid) and 12490 (granulated)).

As used herein, a “viability marker” is a cell characteristic that isindicative of cell viability and is detectable by FACS. Exemplaryviability markers include forward scatter, side scatter, propidiumiodide stain, or combinations thereof.

As used herein, the term “non-AUG start codon” is intended to includeany non-AUG polynucleotide (typically a triplet) that functions as astart site for translation initiation with reduced efficiency relativeto that of an AUG start codon. Naturally occurring alternate start codonusage is known in the art and described for example in Kozak (1991) J.Cell Biol. 115(4): 887-903; Mehdi et al. (1990) Gene 91:173-178; Kozak(1989) Mol. Cell. Biol. 9(11): 5073-5080. In general, non-AUG startcodons have decreased translation efficiencies compared to that of anAUG; for example, the alternate start codon GUG may have 3-5%translation efficiency compared to that of an AUG (100%). Thetranslation efficiency of a non-AUG start codon can also be affected byits sequence context; for example, an optimal Kozak consensus sequenceis reported to have a positive effect on translation initiation atnon-AUG start codons (Mehdi et al. (1990) Gene 91:173-178; Kozak (1989)Mol. Cell. Biol. 9(11): 5073-5080). The complete Kozak DNA consensussequence is GCCRCCATGG (SEQ ID NO:1), where the start codon ATG (AUG inRNA) is bold, the A of the ATG start codon is designated as the +1position, and “R” at position −3 is a purine (A or G). The two mosthighly conserved positions are a purine, preferably an A, at −3 and a Gat +4 (Kozak (1991) J Cell Biol 115(4): 887-903). Alternate start codonusage is described for attenuated expression of a selectable marker inU.S. Patent Publication 2006/0172382 and U.S. Patent Publication2006/0141577, the entire contents of which are incorporated herein byreference. One of skill in the art will recognize that the sequencesdescribed herein as DNA will have correlative sequences as RNAmolecules, e.g., DNA sequence ATG would correspond to RNA sequence AUG,and vice versa.

As used herein, the terms “rapid bulk sorting” and “rapid bulking” referto methods of fluorescence-activated cell sorting (FACS) to batch selectproducer cells expressing a target polypeptide. The methods comprise thesteps of (a) providing a heterogeneous population of producer cells,wherein the producer cells in the population express varying levels of aFACS selectable polypeptide and a target polypeptide that are encoded bythe same multicistronic mRNA; (b) selecting from the heterogeneouspopulation of producer cells a first heterogeneous sub-population ofproducer cells using FACS, wherein the producer cells in the firstheterogeneous sub-population express the FACS selectable polypeptide ata level that is higher than the level of at least 80% of the producercells in the heterogeneous population in (a); and (c) expanding thefirst heterogeneous sub-population of producer cells indrug-selection-free medium, thereby producing an expanded firstheterogeneous sub-population of producer cells.

The rapid bulking method may further comprise the steps of (d) selectingfrom the expanded first heterogeneous sub-population of producer cellsin step (c) a second heterogeneous sub-population of producer cellsusing FACS, wherein the producer cells in the second sub-populationexpress the FACS selectable polypeptide at a level that is higher thanthe level of at least 80% of the producer cells in the expanded firstheterogeneous sub-population of producer cells in step (c); and (e)expanding the second heterogeneous sub-population of producer cells in adrug-selection-free medium, thereby producing an expanded secondheterogeneous sub-population of producer cells.

As used herein, the term “EPIC” refers to Early Post-transfectionIsolation of Cells, as described in more detail herein.

As used herein, the term “FLARE” refers to “FLow cytometry AttenuatedReporter Expression.” FLARE is an expression system utilizing amulticistronic mRNA that contains at least two open reading frames(ORFs), an upstream ORF containing a non-AUG start codon and encoding aFACS selectable polypeptide, and a downstream ORF containing an AUGstart codon and encoding a target polypeptide. See US Patent ApplicationPublication No. 2009/0239235 which is incorporated by reference hereinin its entirety.

As used herein, the term “about” shall refer to a range of tolerance of10% around a stated value. Therefore, when the term “about” is used tomodify a stated value, the range indicated will encompass any numberwithin ±0.01%, 0.02%, 0.05%, 0.1%, 0.2%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%,7%, 8%, 9%, or 10% of the stated value.

II. Methods for Early Selection of Producer Cells

In some aspects, the disclosure relates to a method of producing apopulation of producer cells expressing a target polypeptide. In someembodiments, the method comprises:

-   -   (a) transfecting host cells with one or more vectors that encode        one or more mRNAs, wherein the one or more mRNAs encode a        selectable polypeptide and the target polypeptide;    -   (b) isolating from the transfected host cells, within 2 to 15        days of transfection, a sub-population of early-expressing        transfected host cells which express the selectable polypeptide;        and    -   (c) expanding the sub-population of transfected host cells,        thereby producing a population of producer cells expressing the        target polypeptide.

Early-expressing transfected cells can comprise different classes ofexogenous DNA, part of which has not integrated into the cells' genomicDNA, and part of which has integrated into the cells' genomic DNA. Boththese types of DNA have the potential to lead to expression of thepolypeptide or polypeptides they encode.

Host cells are transfected with one or more vectors that encode one ormore mRNAs, wherein the one or more mRNAs encode a selectablepolypeptide and the target polypeptide. A producer cell can be generatedusing any cell type suitable for production of a target polypeptide froma multicistronic mRNA. In some embodiments, the host cell is aeukaryotic cell. Examples of suitable eukaryotic cells to produce atarget polypeptide include, but are not limited to, a Chinese HamsterOvary (CHO) cell line, including those designated CHO-DBX11, CHO-DG44,CHO-S, CHO-K1, and the hamster cell line BHK-21; the murine cell linesdesignated NIH3T3, NS0, C127, the simian cell lines COS, Vero; and thehuman cell lines HeLa, HEK293 (also called 293), NIH-3T3, U-937 and HepG2. Additional examples of suitable host cells include yeast cells,insect cells (e.g., Drosophila Schneider S2 cells, 519 insect cells (WO94/26087), BTI-TN-5B1-4 (High Five™) insect cells (Invitrogen)), plantcells, avian cells, and bovine cells. Examples of yeast useful forexpression include, but are not limited to Saccharomyces,Schizosaccharomyces, Hansenula, Candida, Torulopsis, Yarrowia, andPichia. See e.g., U.S. Pat. Nos. 4,812,405; 4,818,700; 4,929,555;5,736,383; 5,955,349; 5,888,768 and 6,258,559. Other examples ofproducer cells can be prokaryotic, including bacterial cells such as E.coli (e.g., strain DH5α™) (Invitrogen, Carlsbad, CA), PerC6 (Crucell,Leiden, NL), B. subtilis and/or other suitable bacteria. The cells canbe purchased from a commercial vendor such as the American Type CultureCollection (ATCC, Rockville, MD) or cultured from an isolate usingmethods known in the art.

To make a producer cell, recombinant or exogenous polynucleotide(s) canbe inserted into the host cell using any suitable transfer technique(e.g., by transfection, transformation, electroporation ortransduction). Vectors that encode one or more mRNAs include DNAvectors. Vectors that may be used include plasmids, viruses, phage,transposons, and minichromosomes of which plasmids are a typicalembodiment. Generally such vectors further include a signal sequence,origin of replication, one or more marker genes, a promoter andtranscription termination sequences operably linked to the gene encodingthe multicistronic mRNA so as to facilitate expression. Examples ofsuitable DNA viral vectors include adenovirus (Ad) and adeno-associatedvirus (AAV). Adenovirus-based vectors for the delivery ofpolynucleotides are known in the art and may be obtained commercially orconstructed by standard molecular biological methods. Adenoviruses (Ads)are a group of viruses, including over 50 serotypes. See, e.g.,International Patent Application No. WO 95/27071. Other viral vectorsfor use in the present disclosure include vectors derived from vaccinia,herpesvirus (e.g., herpes simplex virus (HSV)), and retroviruses. Genedelivery vehicles also include several non-viral vectors, includingDNA/liposome complexes, and targeted viral protein-DNA complexes.

For use in transfection, in certain embodiments circular vectors may bepre-linearized, i.e., linearized prior to introduction into the hostcell, for example by restriction at one or more restriction endonucleasesites. Linearization is believed to be necessary for integration intothe genome, and this can be effected by pre-linearization or in a randomfashion by endonucleases naturally present within the host cell.Pre-linearization has the potential advantage of introducing a degree ofcontrol into the site of restriction. Thus, in certain embodiments,circular vectors, including supercoiled circular vectors, may beintroduced into the host cell. In certain embodiments in accordance withthe instant invention, the one or more vectors are linear at the time oftransfection.

Vectors that contain both a promoter and a cloning site into which apolynucleotide can be operatively linked are known in the art andavailable from commercial vendors. Such vectors are capable oftranscribing RNA in vitro or in vivo, and are commercially availablefrom sources such as Agilent Technologies and Promega Corporation. Inorder to optimize expression and/or in vitro transcription, it may benecessary to remove, add or alter 5′ and/or 3′ untranslated portions toeliminate extra, potentially inappropriate alternative translationinitiation codons or other sequences that may interfere with or reduceexpression, either at the level of transcription or translation.Alternatively, consensus ribosome binding sites can be insertedimmediately 5′ of the start codon to enhance expression.

A promoter can be provided for expression in the producer cell.Promoters can be constitutive or inducible. For example, a promoter canbe operably linked to a nucleic acid encoding a multicistronic mRNA suchthat it directs expression of the encoded polypeptides. A variety ofsuitable promoters for prokaryotic and eukaryotic hosts are available.Prokaryotic promoters include lac, tac, T3, T7 promoters for E. coli;3-phosphoglycerate kinase or other glycolytic enzymes e.g., enolase,glyceraldehyde 3-phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, glucose 6 phosphate isomerase,3-phosphoglycerate mutase, and glucokinase. Eukaryotic promoters includeinducible yeast promoters such as alcohol dehydrogenase 2, isocytochromeC, acid phosphatase, metallothionein, and enzymes responsible fornitrogen metabolism or maltose/galactose utilization; RNA polymerase IIpromoters including viral promoters such as polyoma, fowlpox andadenoviruses (e.g., adenovirus 2), bovine papilloma virus, avian sarcomavirus, cytomegalovirus (CMV, in particular, the immediate early genepromoter), retrovirus, hepatitis B virus, actin, Rous sarcoma virus(RSV) promoter, and the early or late Simian virus 40 (SV40) andnon-viral promoters such as EF-1 alpha (Mizushima and Nagata (1990)Nucleic Acids Res. 18(17):5322). Those of skill in the art will be ableto select the appropriate promoter for expressing any given polypeptidein a given host cell.

Where appropriate, e.g., for expression in cells of higher eukaryotes,additional enhancer elements can be included instead of or as well asthose found located in the promoters described above. Suitable mammalianenhancer sequences include enhancer elements from globin, elastase,albumin, fetoprotein, metallothionein, and insulin. Alternatively, onemay use an enhancer element from a eukaryotic cell virus such as SV40enhancer, cytomegalovirus early promoter enhancer, polyoma enhancer,baculoviral enhancer or murine IgG2a locus (see, WO 2004/009823). Whilstsuch enhancers are often located on the vector at a site upstream to thepromoter, they can also be located elsewhere, e.g., within theuntranslated region or downstream of the polyadenylation signal. Thechoice and positioning of enhancer may be based upon compatibility withthe host cell used for expression.

In addition, the vectors (e.g., expression vectors) may comprise aselectable marker for selection of host cells carrying the vector, and,in the case of a replicable vector, an origin of replication. Genesencoding products which confer antibiotic or drug resistance are commonselectable markers and may be used in prokaryotic (e.g., f3-lactamasegene (ampicillin resistance), tet gene (tetracycline resistance) andeukaryotic cells (e.g., neomycin (G418 or geneticin), gpt (mycophenolicacid), ampicillin, or hygromycin B resistance genes). The dihydrofolatereductase (DHFR) gene permits selection with methotrexate ornucleotide-deficient medium in a variety of hosts. Similarly, theglutamine synthetase (GS) gene permits selection with methioninesulphoximine. Genes encoding the gene product of auxotrophic markers ofthe host (e.g., LEU2, URA3, HIS3) are often used as selectable markersin yeast. Use of viral (e.g., baculovirus) or phage vectors, and vectorswhich are capable of integrating into the genome of the host cell, suchas retroviral vectors, are also contemplated.

In eukaryotic systems, polyadenylation and termination signals may beoperably linked to a polynucleotide encoding the multicistronic mRNA asdescribed herein. Such signals are typically placed 3′ of an openreading frame. In mammalian systems, non-limiting examples ofpolyadenylation/termination signals include those derived from growthhormones, elongation factor-1 alpha and viral (e.g., SV40) genes orretroviral long terminal repeats. In yeast systems, non-limitingexamples of polyadenylation/termination signals include those derivedfrom the phosphoglycerate kinase (PGK) and the alcohol dehydrogenase 1(ADH) genes. In prokaryotic systems polyadenylation signals aretypically not required and it is instead usual to employ shorter andmore defined terminator sequences. The choice ofpolyadenylation/termination sequences may be based upon compatibilitywith the host cell used for expression. In addition to the above, otherfeatures that can be employed to enhance yields include chromatinremodeling elements, introns and host cell specific codon modification.

The producer cells of the disclosure contain a recombinantpolynucleotide (e.g., a recombinant cDNA) that encodes a multicistronicmRNA molecule from which the target and selectable polypeptides areseparately translated from different ORFs. In some embodiments, theselectable polypeptide is a cell surface polypeptide. In certainembodiments, the producer cells of the disclosure contain a plurality ofrecombinant polynucleotides, each of which encodes a multicistronic mRNAmolecule from which a target polypeptide and a selectable polypeptideare separately translated from different ORFs. Each target polypeptidecan thus be associated with a particular selectable polypeptide. In someembodiments, the selectable polypeptide is a cell surface polypeptide.

Examples of cell surface polypeptides include, but are not limited toCD2, CD20, CD52, and CD59. Exemplary, non-limiting, amino acid sequencesfor CD52 and CD59 cell surface polypeptides are provided below.

Amino Acid Sequence for Exemplary Human CD52 polypeptide: (SEQ ID NO: 2)LERFLFLLLTISLLVLVQIQTGLSGQNDTSQTSSPSASSNISGGIFLFFV ANAIIHLFCFSAmino Acid Sequence for Exemplary HumanCD59 polypeptide (splice acceptor mutant): (SEQ ID NO: 3)LGIQGGSVLFGLLLVLAVFCHSGHSLQCYNCPNPTADCKTAVNCSSDFDACLITKAGLQVYNNCWKFEHCNFNDVTTRLRENELTYYCCKKDLCNFNEQLENGGTSLSEKTVLLLVTPFLAAAWSLHP Amino Acid Sequence for Exemplary MouseCD52 polypeptide: (SEQ ID NO: 4)LKSFLLFLTIILLVVIQIQTGSLGQATTAASGTNKNSTSTKKTPLKSGASSIIDAGACSFLFFANTLICLFYLS

In some embodiments, a first ORF is provided which encodes a selectablepolypeptide, such as CD52 or CD59. Exemplary, non-limiting ORF sequencesfor CD52 and CD59 are provided below.

Nucleotide Sequence for Exemplary Human CD52 ORF: (SEQ ID NO: 5)ttggagcgcttcctcttcctcctactcaccatcagcctcctcgttttggtacaaatacaaaccggactctccggacaaaacgacaccagccaaaccagcagcccctcagcatccagcaacataagcggaggcattttccttttcttcgtcgccaacgccataatccacctcttctgcttcagttgaNucleotide Sequence for Exemplary Human CD59 ORF: (SEQ ID NO: 6)ttgggaatccaaggagggtctgtcctgttcgggctgctgctcgtcctcgctgtcttctgccattccggtcatagcctgcagtgctacaactgtcctaacccaactgctgactgcaaaacagccgtcaattgttcatctgattttgacgcgtgtctcattaccaaagctgggttacaagtgtataacaactgttggaagtttgagcattgcaatttcaacgacgtcacaacccgcttgagggaaaacgagctaacgtactactgctgcaagaaggacctgtgtaactttaacgaacagcttgaaaacggagggacatccttatcagagaaaacagttcttctgctggtgactccatttctggcagctgcttggagccttcatccctaaNucleotide Sequence for Exemplary Mouse CD52 ORF: (SEQ ID NO: 7)ttgaagagcttcctcctcttcctcactatcattcttctcgtagtcattcagatacaaacaggatccttaggacaagccactacggccgcttcaggtactaacaaaaacagcacctccaccaaaaaaacccccttaaagagcggggcctcatccatcatcgacgcgggcgcttgcagtttcctcttcttcgccaatacccttatttgcctcttctacctcagctaactgagtaa

As discussed below, each the foregoing exemplary ORFs has been modifiedto eliminate all internal ATG triplets.

In some embodiments, a second ORF is provided which encodes a targetpolypeptide, such as an antibody, enzyme, or Fc fusion protein. In someembodiments, separate translation is accomplished by use of a non-AUGstart codon for translation initiation of the selectable polypeptide andthe use of an AUG start codon for translation initiation of the targetpolypeptide. In this embodiment, generally the polynucleotide encodingthe target polypeptide is located downstream of the polynucleotideencoding the selectable polypeptide. Separate translation can also beachieved using an internal ribosome entry site (IRES). In someembodiments, the IRES element is located upstream of the polynucleotideencoding the target polypeptide and downstream of the polynucleotideencoding the selectable polypeptide. In some embodiments, the IRESelement is located upstream of the polynucleotide encoding theselectable polypeptide and downstream of the polynucleotide encoding thetarget polypeptide.

In some embodiments, a non-AUG start codon is located within the DNAencoding the selectable polypeptide in such a way that translation ofthe selectable polypeptide is less efficient than translation of thetarget polypeptide. To achieve decreased translation efficiency, the AUGstart codon of the selectable polypeptide may be changed to an alternatenon-AUG start codon, examples of which include but are not limited to:CUG, GUG, UUG, AUU, AUA, and ACG.

Thus, when using an alternate non-AUG start codon, expression of aselectable polypeptide can be attenuated relative to that of aco-expressed target polypeptide. In addition to alteration of the startcodon, the DNA encoding the selectable polypeptide may be modified atall internal ATG triplets to prevent internal initiation of translation.In some embodiments, the selectable polypeptide has a short amino acidsequence (<200 amino acids) and is encoded by a polynucleotide with few(<10) ATG triplets.

Without wishing to be bound by theory, to initiate translation of themRNA encoding both the selectable polypeptide and the targetpolypeptide, ribosomes begin scanning at the 5′ cap structure of themRNA with the majority scanning past the alternate start codon (forexample, UUG) and instead initiating translation at the downstream AUGstart codon. However, translation initiation can occur at the alternatestart codon, albeit with very low frequency, so that a low level of theselectable polypeptide is also expressed.

From the transfected host cells is selected a sub-population ofearly-expressing transfected host cells which express detectable levelsof the selectable polypeptide. During transfection individual host cellstake up different amounts of exogenous polynucleotide, e.g., DNA, in anessentially random manner. Some cells will take up many copies of theexogenous polynucleotide, others will take up fewer copies, and somewill take up none. The amount of DNA taken up into a given cell affectsthe fate of the DNA, including its early expression and its integrationinto the genome.

Following transfection with DNA, at least some of the polynucleotidethat has been introduced into the cell is translocated into the nucleuswhere it is transcribed into mRNA. In the first few days, expression ofthe introduced DNA may be driven off one or more classes of DNA, some ofwhich has not yet been integrated into the genome of the host cell, andsome of which has been integrated into the genome of the host cell. Atthis point the extent of expression is believed to be principallyproportional to the “dose” of DNA introduced into the host cell and itsnucleus. The greater the amount of exogenous DNA taken up by the hostcell the greater the degree of early expression. However, a small amountof DNA that has been introduced into the host cell, particularly once itis linear, can become integrated into the genome of the host cell. Thus,in the first several days following transfection, there is a competitionbetween degradation and loss of the exogenous DNA on the one hand, andstochastic integration of the exogenous DNA into the genome on the otherhand. Integration can include single or multiple copies of introducedDNA. The greater the amount of exogenous DNA taken up by the host cell,the greater the chance (and degree) of its integration. Ultimately, itis the integrated DNA that is responsible for long-term productiveexpression, i.e., expression after all nonintegrated DNA (e.g., plasmidor episomal DNA) is degraded to the point of being incapable ofmeaningful expression.

Thus in the first 2 to about 15 days (e.g., 2 days, 3 days, 4 days, 5days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13days, 14 days, 15 days) following transfection, there areearly-expressing transfected host cells which express detectable amountsof the selectable polypeptide. Particularly in the first 2-6 days, moreparticularly in the first 2-4 days, and even more particularly in thefirst 2-3 days, this early expression is believed to be largely, but notnecessarily exclusively, driven off exogenous DNA that has not yet beenintegrated into the genome of the host cell. During this early periodfollowing transfection, there may be some degree of integration ofexogenous DNA into the host cell genome. Because this early expressiondepends on the “dose” of DNA taken up by the host cell and its nucleus,and the dose is essentially random among transfected cells, during thisearly period the transfected host cells include sub-populations of cellsexpressing different amounts of polypeptide encoded by the exogenousDNA. Also during this early period the sub-populations of cellsexpressing greater amounts of polypeptide encoded by the exogenous DNApresumably took up greater amounts of exogenous DNA and therefore have agreater chance of incorporating the DNA into their genome.

Accordingly, the term “early-expressing” or “early expression”, as usedherein, refers to detectable expression in the first 2 to about 15 days(e.g., 2-15 days, 2-14 days, 2-13 days, 2-12 days, 2-11 days, 2-10 days,2-9 days, 2-8 days, 2-7 days, 2-6 days, 2-5 days, 2-4 days, 2-3 days,3-15 days, 3-14 days, 3-13 days, 3-12 days, 3-11 days, 3-10 days, 3-9days, 3-8 days, 3-7 days, 3-6 days, 3-5 days, 3-4 days, 4-15 days, 4-14days, 4-13 days, 4-12 days, 4-11 days, 4-10 days, 4-9 days, 4-8 days,4-7 days, 4-6 days, 4-5 days, 5-15 days, 5-14 days, 5-13 days, 5-12days, 5-11 days, 5-10 days, 5-9 days, 5-8 days, 5-7 days, 5-6 days, 6-15days, 6-14 days, 6-13 days, 6-12 days, 6-11 days, 6-10 days, 6-9 days,6-8 days, 6-7 days, 7-15 days, 7-14 days, 7-13 days, 7-12 days, 7-11days, 7-10 days, 7-9 days, 7-8 days, 8-15 days, 8-14 days, 8-13 days,8-12 days, 8-11 days, 8-10 days, 8-9 days, 9-15 days, 9-14 days, 9-13days, 9-12 days, 9-11 days, 9-10 days) following transfection. Incertain embodiments, the term “early-expressing” or “early expression”refers to detectable expression in the first 2 to about 10 daysfollowing transfection. In certain embodiments, the term“early-expressing” or “early expression” refers to detectable expressionin the first 2 to about 6 days following transfection. In certainembodiments, the term “early-expressing” or “early expression” refers todetectable expression in the first 2 to about 5 days followingtransfection. In certain embodiments, the term “early-expressing” or“early expression” refers to detectable expression in the first 2 toabout 4 days following transfection. In certain embodiments, the term“early-expressing” or “early expression” refers to detectable expressionin the first 2 to about 3 days following transfection.

Any method known in the art useful for detecting a cell surface markermay be used in connection with the methods of the disclosure. Forexample, an antibody or other cell surface marker-specific binding agentis contacted directly or indirectly with the transfected host cellsunder conditions that permit or favor binding of antibody to theselectable polypeptide and thereby select a sub-population ofearly-expressing transfected host cells. The selection of the antibodyor other binding agent is determined by: 1) its ability to selectivelybind the selectable polypeptide that is expressed on the host cell; and2) its ability to be labeled with a detectable label or bind to adetectable label, for example, for use in flow cytometry or FACS.

In an alternate embodiment, a first agent can be a protein or peptidethat binds to the selectable polypeptide, which first agent also in turnbinds to a second agent that is capable of being detectably labeled(e.g., incorporating a fluorescent, enzymatic, colorimetric,magnetically susceptible, or other detectable label). It is intended,although not always explicitly stated that “indirect” binding to theselectable polypeptide includes the use of any number of intermediatepartners. In certain embodiments, “indirect” binding to the selectablepolypeptide includes the use of one intermediate partner, e.g., oneunlabeled antibody or other binding agent.

In some embodiments, the antibody or other binding agent binds directlyto the cell surface marker and comprises a fluorescent label. Suitablefluorescent labels include, but are not limited to, fluoresceinisothiocyanate (FITC), rhodamine, tetramethylrhodamine, eosin,phycoerythrin (PE), erythrosin, allophycocyanin (APE), coumarin,methyl-coumarins, pyrene, Malachite green, stilbene, Lucifer Yellow,Cascade Blue, and Texas Red. Other suitable optical dyes are describedin the Molecular Probes® Handbook, 11^(th) Edition, 2010.

In some embodiments, the fluorescent label is functionalized tofacilitate covalent attachment to the antibody or other agent. Suitablefunctional groups, include, but are not limited to, isothiocyanategroups, amino groups, haloacetyl groups, maleimides, succinimidylesters, and sulfonyl halides, all of which may be used to attach thefluorescent label to a second molecule. The choice of the functionalgroup of the fluorescent label will depend on the site of attachment tothe antibody or other binding agent, the selectable polypeptide, or thesecond labeling agent.

Attachment of the fluorescent label may be either direct or via a linkerto the antibody or other binding agent. In one aspect, the linker is arelatively short coupling moiety that generally is used to attachmolecules. In this embodiment, attachment of the first labeling moietyto the candidate agents will be done as is generally appreciated bythose in the art, and may include techniques outlined above for theincorporation of fluorescent labels.

Materials and techniques for design and construction of labeledantibodies and other agents for use in cytometry are known in the artand described for example, in Bailey et al. (2002) Biotechnol. Bioeng.80(6); 670-676; Carroll and Al-Rubeai (2004) Expt. Opin. Biol. Therapy4:1821-1829; Yoshikawa et al. (2001) Biotechnol. Bioeng. 74:435-442;Meng et al. (2000) Gene 242:201-207; Borth et al. (2001) Biotechnol.Bioeng. 71 (4):266-273; Zeyda et al. (1999) Biotechnol. Prog.15:953-957; Klucher et al. (1997) Nucleic Acids Res. 25(23):4853-4860;and Brezinsky et al. (2003) J. Imumunol. Methods 277:141-155.

Suitable binding pairs for use in indirectly linking the label to theagent (which in turn, binds the selectable polypeptide) include, but arenot limited to, antigens/antibodies, including digoxigenin/antibody,dinitrophenol (DNP)/anti-DNP, dansyl-X/anti-dansyl,fluorescein/anti-fluorescein, lucifer yellow/anti-lucifer yellow,rhodamine/anti-rhodamine; and biotin/avidin (or biotin/streptavidin).The binding pairs should have high affinities for each other, sufficientto withstand the shear forces during cell sorting or other detectionsystem used in connection with the disclosure.

Thus, in some aspects, first labeling moieties (when second labelingmoieties are used), include, but are not limited to, haptens such asbiotin. Biotinylation of target molecules is well known, for example, alarge number of biotinylation agents are known, including amine-reactiveand thiol-reactive agents, for the biotinylation of proteins, nucleicacids, carbohydrates, and carboxylic acids. Similarly, a large number ofother haptenylation reagents are also known.

The antibodies used in a method described herein can be produced in cellculture, in phage, or in various animals, including but not limited tomice, rats, hamsters, guinea pigs, rabbits, sheep, goats, horses, cows,camelids, monkeys, chimpanzees, etc., so long as the antibodies retainspecificity of binding for the selectable polypeptide. Antibodies can betested for specificity of binding by comparing binding to appropriateantigen to binding to irrelevant antigen or antigen mixture under agiven set of conditions.

In embodiments in which the antibody or other binding agent for theselectable polypeptide is not directly labeled, the antibody or bindingagent preferably also contains and retains the ability to bind asecondary agent which is detectable after binding to the cell via theselectable polypeptide.

In some embodiments, when the selectable polypeptide is CD52, theselectable polypeptide may be detected using an anti-CD52 antibody.“Anti-CD52 antibody” refers to an antibody that specifically recognizesand binds CD52. Anti-CD52 antibodies can be generated by methods wellknown in the art. See for example, Current Protocols in MolecularBiology (F. M. Ausubel, et al. eds., 1987 to present versions) andAntibodies: A Laboratory Manual, Second edition (Greenfield, ed. 2013).Additionally, several anti-CD52 antibodies are commercially available(e.g., antibodies conjugated to a fluorescent label, such as those soldby the commercial vendors AbCam, SeroTec, and BioLegend).

In some embodiments, when the selectable polypeptide is CD59, theselectable polypeptide may be detected using an anti-CD59 antibody.“Anti-CD59 antibody” refers to an antibody that specifically recognizesand binds CD59. Anti-CD59 antibodies can be generated by methods wellknown in the art. Additionally, several anti-CD59 antibodies arecommercially available (e.g., antibodies conjugated to a fluorescentlabel, such as those sold by the commercial vendors AbCam, SeroTec, andBioLegend).

In a particular embodiment, when the selectable polypeptide is CD20, theselectable polypeptide may be detected using an anti-CD20 antibody.“Anti-CD20 antibody” refers to an antibody that specifically recognizesand binds CD20. Anti-CD20 antibodies can be generated by methods wellknown in the art. Additionally, several anti-CD20 antibodies arecommercially available from vendors such as BD Pharmingen; BeckmanCoulter, Inc. (Fullerton, Calif, numerous clones including Catalog No.6604106 Clone H299 (B1); Isotype IgG2a and Catalog No. IM1565 Clone L26,Isotype IgG2a); Invitrogen (Carlsbad, Calif., Clone: BH-20, Isotype:IgG2a and Clone: B-H20, Isotype: IgG2a); BioLegend (San Diego, Calif.,Catalog. No. 302301, Clone: 21-7, Isotype: IgG2b, κ); EMD Biosciences,Inc., CALBIOCHEM® Brand (San Diego, Calif, Catalog No. 217670 Clone 2H7,Isotype: IgG2b); and Anaspec (San Jose, Calif, Catalog No. 29587).

For use in MACS, where there is a more limited number ofantigen-specific magnetic beads, a labeled or unlabeled primary antibodyor other binding agent (e.g., Fc fusion protein) can be used to bind tothe selectable polypeptide, followed by binding by, for example, anisotype-specific magnetic bead. For example, Miltenyi Biotec sells CD20microbeads and anti-mouse IgG microbeads, but neither CD52 nor CD59microbeads; anti-mouse IgG microbeads could be used to label primarymouse IgG anti-human CD52 or mouse IgG anti-human CD59.

In an exemplary, non-limiting method, a population of transfected hostcells as described herein is contacted with an agent that recognizes anddirectly or indirectly binds the selectable polypeptide, if present, onthe surface of the cells. The contacting is performed under conditionsthat favor or are suitable for specific binding (directly or indirectly)of the agent or antibody with the selectable polypeptide. The cells thatare bound to the agent or antibody are then selected for using asuitable method such as FACS (e.g., by gating for cells that express theFACS-selectable polypeptide at a high level such as a level that is atleast 80% of the level of the population) and used to select asub-population of early-expressing transfected host cells.Alternatively, the cells that are bound to the agent or antibody arethen selected for using a suitable method such as MACS (e.g., by gatingfor cells that express the MACS-selectable polypeptide at a high levelsuch as a level that is at least 80% of the level of the population) andused to select a sub-population of early-expressing transfected hostcells.

The selected sub-population of early-expressing transfected host cellsis then grown under conditions that result in expansion of thesub-population to produce a population of producer cells expressing thetarget polypeptide.

In certain embodiments, the step of isolating from the transfected hostcells, within 2 to days of transfection, a sub-population ofearly-expressing transfected host cells which express the selectablepolypeptide is performed in drug-selection-free medium. For example, incertain embodiments, the step of isolating from the transfected hostcells, within 2 to 15 days of transfection, a sub-population ofearly-expressing transfected host cells which express the selectablepolypeptide is performed in 0 nM MTX (i.e., MTX-free) medium.

In certain embodiments, the step of expanding the selectedsub-population of transfected host cells is performed indrug-selection-free medium. For example, in certain embodiments, thestep of expanding the selected sub-population of transfected host cellsis performed in 0 nM MTX (i.e., MTX-free) medium.

In certain embodiments, both the step of (b) isolating from thetransfected host cells, within 2 to 15 days of transfection, asub-population of early-expressing transfected host cells which expressthe selectable polypeptide, and the step of (c) expanding the isolatedsub-population of transfected host cells are performed indrug-selection-free medium. For example, in certain embodiments, boththe step of (b) isolating from the transfected host cells, within 2 todays of transfection, a sub-population of early-expressing transfectedhost cells which express the selectable polypeptide, and the step of (c)expanding the isolated sub-population of transfected host cells areperformed in 0 nM MTX (i.e., MTX-free) medium.

Cells, including producer cells, may be cultured in spinner flasks,shake flasks, roller bottles, wave reactors (e.g., System 1000 fromwavebiotech.com) or hollow fiber systems, or for large scale production,stirred tank reactors or bag reactors (e.g., Wave Biotech, Somerset, NewJersey USA) are used particularly for suspension cultures. Stirred tankreactors can be adapted for aeration using e.g., spargers, baffles orlow shear impellers. For bubble columns and airlift reactors, directaeration with air or oxygen bubbles may be used. Where the host cellsare cultured in a serum-free culture medium, the medium can besupplemented with a cell protective agent such as poloxamer 188(Pluronic® F-68) to help prevent cell damage as a result of the aerationprocess. Depending on the host cell characteristics, microcarriers maybe used as growth substrates for anchorage-dependent cell lines, or thecells may be adapted to suspension culture. The culturing of host cells,particularly vertebrate host cells, may utilize a variety of operationalmodes such as batch, fed-batch, repeated batch processing (see, Drapeauet al. (1994) Cytotechnology 15:103-109), extended batch process orperfusion culture. Although recombinantly transformed producer cells maybe cultured in serum-containing media such media comprising fetal calfserum (FCS), in some embodiments, such host cells are cultured inserum-free media such as disclosed in Keen et al. (1995) Cytotechnology17:153-163, or commercially available media such as ProCHO-CDM orUltraCHO™ (Cambrex, NJ, USA), supplemented where necessary with anenergy source such as glucose and synthetic growth factors such asrecombinant insulin. The serum-free culturing of host cells may requirethat those cells are adapted to grow in serum-free conditions. Oneadaptation approach is to culture such host cells in serum containingmedia and repeatedly exchange 80% of the culture medium for theserum-free media so that the host cells adapt to serum-free conditions(see, e.g., Scharfenberg, K. et al. (1995) In: Animal Cell Technology:Developments Towards the 21st Century (Beuvery, E. C. et al., eds), pp.619-623, Kluwer Academic publishers).

In certain embodiments, the method further comprises isolating thetarget polypeptide from the population of producer cells. The targetpolypeptide can be isolated using any method known in the art and may befurther purified, e.g., according to Current Good Manufacturing Practice(CGMP) for recombinant proteins and antibodies, to a purity level of atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, at least 99.5%, or more. A target polypeptide according tothe described embodiments may be secreted into the medium and recoveredand purified therefrom using any of a variety of techniques to provide adegree of purification suitable for the intended use. For example, theuse of a target polypeptide (e.g., an antibody or Fc fusion protein) forthe treatment of human subjects typically mandates at least 95% purityas determined by reducing SDS-PAGE, more typically 98% or 99% purity,when compared to the culture media comprising the target polypeptide. Inthe first instance, cell debris from the culture media can be removedusing centrifugation followed by a clarification step of the supernatantusing e.g., microfiltration, ultrafiltration and/or depth filtration.Alternatively, a target polypeptide can be harvested by microfiltration,ultrafiltration or depth filtration without prior centrifugation. Avariety of other techniques such as dialysis and gel electrophoresis andchromatographic techniques such as hydroxyapatite (HA), affinitychromatography (optionally involving an affinity tagging system such aspolyhistidine) and/or hydrophobic interaction chromatography (HIC) (see,U.S. Pat. No. 5,429,746) are available. In one embodiment, a targetpolypeptide such as an antibody or Fc fusion protein, following variousclarification steps, is captured using Protein A or G affinitychromatography followed by further chromatography steps such as ionexchange and/or HA chromatography, anion or cation exchange, sizeexclusion chromatography and ammonium sulphate precipitation. Variousvirus removal steps may also be employed (e.g., nanofiltration using,e.g., a DV-20 filter). Following these various steps, a purifiedpreparation comprising at least 10 mg/mL or greater, e.g., 100 mg/mL orgreater of the target polypeptide described herein is provided.

In certain embodiments the methods of the invention further include thestep of isolating one or more single transfected host cells from theexpanded sub-population and culturing the one or more single transfectedhost cells to produce clonal populations of the one or more singletransfected host cells. In certain embodiments the methods of theinvention further include the step of isolating one or more singletransfected host cells from the expanded sub-population and culturingthe one or more single transfected host cells to produce one or moreclonal populations of producer cells expressing the target polypeptide.Preparation of a clonal population can be performed by any method knownin the art. For example, in one embodiment, the selected cells may beplated into 96-well (or other size) plates at a density of one cell perwell and permitted to grow for a period of time (e.g., typically 7-28days) which permits the single cell to grow into a multi-cell colony ofdaughter cells (i.e., a clonal population). The method may next compriseanalyzing one or more of the clonal populations by detecting the levelof the selectable polypeptide and/or target polypeptide expression onsaid clonal population and selecting one or more clonal populations witha high expression level of the selectable polypeptide and/or targetpolypeptide, thereby selecting one or more clonal populations stablyexpressing the target polypeptide. In certain embodiments, the clonalpopulation is cultured for 7-28 days after plating at a single celldensity before the clonal populations are analyzed. The method mayfurther include contacting the clonal population with a detectableantibody or other binding agent that recognizes and directly orindirectly binds the selectable polypeptide, if present, on the surfaceof the clonal cell under conditions that permit or favor binding of theantibody or other binding agent with the selectable polypeptide; andselecting or detecting one or more cells that are directly or indirectlybound to the antibody or other binding agent. These cells so selectedalso can be isolated and cultured. The method may further includeanalyzing target polypeptide expression of the one or more clones, e.g.,using protein A screening (such as when the target polypeptide is anantibody or Fc fusion protein), Western blot, SDS polyacrylamide gelelectrophoresis (PAGE) with Coomassie Blue or silver stain, or an enzymeactivity assay.

In certain embodiments, the sub-population of transfected host cellssubject to isolation in step (b) comprises at least 80-120×10⁶ cells.For example, in certain embodiments, the sub-population of transfectedhost cells subject to isolation in step (b) comprises at least about80×10⁶ cells; in certain embodiments, the sub-population of transfectedhost cells subject to isolation in step (b) comprises at least about90×10⁶ cells; in certain embodiments, the sub-population of transfectedhost cells subject to isolation in step (b) comprises at least about100×10⁶ cells; in certain embodiments, the sub-population of transfectedhost cells subject to isolation in step (b) comprises at least about110×10⁶ cells; and in certain embodiments, the sub-population oftransfected host cells subject to isolation in step (b) comprises atleast about 120×10⁶ cells. For example, in certain embodiments, thesub-population of transfected host cells subject to isolation in step(b) comprises about 80×10⁶ to about 800×10⁶ cells, about 100×10⁶ toabout 800×10 6 cells, about 200×10⁶ to about 800×10⁶ cells, about300×10⁶ to about 800×10⁶ cells, about 400×10⁶ to about 800×10⁶ cells,about 500×10⁶ to about 800×10⁶ cells, about 80×10⁶ to about 600×10⁶cells, about 100×10⁶ to about 600×10⁶ cells, about 200×10⁶ to about600×10⁶ cells, about 300×10⁶ to about 600×10⁶ cells, about 400×10⁶ toabout 600×10⁶ cells, about 500×10⁶ to about 600×10⁶ cells, about 80×10⁶to about 500×10⁶ cells, about 100×10 6 to about 500×10⁶ cells, about200×10⁶ to about 500×10⁶ cells, about 300×10⁶ to about 500×10⁶ cells,about 400×10⁶ to about 500×10⁶ cells, about 80×10⁶ to about 400×10⁶cells, about 100×10⁶ to about 400×10⁶ cells, about 200×10⁶ to about400×10⁶ cells, about 300×10⁶ to about 400×10⁶ cells, about 80×10⁶ toabout 300×10⁶ cells, about 100×10⁶ to about 300×10 6 cells, about200×10⁶ to about 300×10⁶ cells, about 80×10⁶ to about 250×10⁶ cells,about 100×10⁶ to about 250×10⁶ cells, about 200×10⁶ to about 250×10⁶cells, about 80×10⁶ to about 200×10⁶ cells, or about 100×10⁶ to about200×10⁶ cells.

In certain embodiments, the isolation in step (b) is performed less than6 days after transfection. For example, in certain embodiments, theisolation in step (b) is performed between two and four days aftertransfection. In certain embodiments, the isolation in step (b) isperformed two days after transfection. In certain embodiments, theisolation in step (b) is performed three days after transfection.

In certain embodiments, the sub-population of transfected host cellscomprises about 0.5-6.0×10⁶ cells prior to expansion in step (c). Forexample, in certain embodiments, the sub-population of transfected hostcells comprises about 0.5×10⁶ cells, about 1.0×10⁶ cells, about 2.0×10⁶cells, about 3.0×10⁶ cells, about 4.0×10⁶ cells, about 5.0×10⁶ cells, orabout 6.0×10⁶ cells prior to expansion in step (c). For example, incertain embodiments, the sub-population of transfected host cellscomprises about 0.5×10⁶ to about 1.0×10⁶ cells, about 0.5×10⁶ to about2.0×10⁶ cells, about 0.5×10⁶ to about 3.0×10⁶ cells, about 0.5×10⁶ toabout 4.0×10 6 cells, about 0.5×10⁶ to about 5.0×10⁶ cells, about0.5×10⁶ to about 6.0×10⁶ cells, about 1.0×10⁶ to about 2.0×10⁶ cells,about 1.0×10⁶ to about 3.0×10⁶ cells, about 1.0×10⁶ to about 4.0×10⁶cells, about 1.0×10⁶ to about 5.0×10⁶ cells, about 1.0×10⁶ to about6.0×10⁶ cells, about 2.0×10⁶ to about 3.0×10⁶ cells, about 2.0×10⁶ toabout 4.0×10⁶ cells, about 2.0×10⁶ to about 5.0×10⁶ cells, about 2.0×10⁶to about 6.0×10⁶ cells, about 3.0×10⁶ to about 4.0×10 6 cells, about3.0×10⁶ to about 5.0×10⁶ cells, about 3.0×10⁶ to about 6.0×10⁶ cells,about 4.0×10⁶ to about 5.0×10⁶ cells, about 4.0×10⁶ to about 6.0×10⁶cells, or about 5.0×10⁶ to about 6.0×10⁶ cells, prior to expansion instep (c). In certain embodiments, the sub-population of transfected hostcells contains greater than 6.0×10⁶ cells prior to expansion in step(c). For example, in certain embodiments, the sub-population oftransfected host cells comprises about 7.0×10⁶ cells, about 8.0×10⁶cells, about 9.0×10⁶ cells, or about 10.0×10⁶ cells, prior to expansionin step (c).

In certain embodiments, the expanding in step (c) is for between 4-31days. For example, in various embodiments, the expanding is for 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, or 31 days.

In certain embodiments, a first of the one or more vectors encodes themRNA encoding the target polypeptide, and a second of the one or morevectors encodes the mRNA encoding the selectable polypeptide. Where theone or more vectors encoding the mRNA encoding the target polypeptideand the mRNA encoding the selectable polypeptide are separate vectors,in certain embodiments the vectors are independently selected fromplasmids, viruses, phage, transposons, and minichromosomes.

In certain embodiments, the mRNA encoding the target polypeptide and themRNA encoding the selectable polypeptide are both encoded on one vector.In accordance with these embodiments, a single vector encodes apolycistronic mRNA encoding both the target polypeptide and theselectable polypeptide. Also in accordance with these embodiments, incertain embodiments the mRNA encoding the selectable polypeptide can beupstream (i.e., 5′) of the mRNA encoding the target polypeptide.Alternatively, in certain embodiments the mRNA encoding the targetpolypeptide can be upstream (i.e., 5′) of the mRNA encoding theselectable polypeptide.

Thus in certain embodiments, the target polypeptide and the selectablepolypeptide are encoded by a single multicistronic mRNA. In certainembodiments, the multicistronic mRNA comprises a first open readingframe (ORF) that encodes the selectable polypeptide and a second ORFthat encodes the target polypeptide, wherein the first ORF is 5′ to thesecond ORF.

In certain embodiments, the first ORF has a non-AUG start codon. Incertain embodiments, the non-AUG start codon is a UUG, GUG, or CUG in aKozak consensus sequence. A non-AUG start codon can be installed usingstandard molecular biology techniques such as are well known in the art.

In certain embodiments, the second ORF has an AUG start codon.

In certain embodiments, the first ORF has a non-AUG start codon, and thesecond ORF has an AUG start codon.

In certain embodiments, the ORF that encodes the selectable polypeptideis devoid of any AUG sequences. AUG sequences can be converted to othertriplet sequences, other than stop codons, using standard molecularbiology techniques such as are well known in the art; for example, andwithout limitation, AUG sequences can be converted independently to CUG(L), GUG (V), UUG (L), AAG (K), ACG (T), AGG (R), AUA (I), AUC (I), AUU(I), GCA (A), GCC (A), GCG (A), or GCU (A).

In certain embodiments, the target polypeptide and the selectablepolypeptide form a fusion protein. In certain embodiments, the fusionprotein is membrane-bound. When the fusion protein is membrane-bound, incertain embodiments the selectable polypeptide is present in adetectable form, i.e., the target polypeptide portion of the fusionprotein does not prohibit detection of the selectable polypeptideportion of the fusion protein. Also when the fusion protein ismembrane-bound, in certain embodiments the target polypeptide is presentin a functional form, i.e., the selectable polypeptide portion of thefusion protein does not prohibit function of the target polypeptideportion of the fusion protein. In certain embodiments, the fusionprotein is released from the host cell as a soluble protein. In certainembodiments, the fusion protein is expressed as a surface protein butcan be cleaved to release the target polypeptide in a soluble,functional form.

In certain embodiments, the target polypeptide is a therapeutic agent,e.g., an antibody, an antigen-binding fragment of an antibody, an Fcfusion protein, a hormone, or an enzyme. Polypeptide hormones include,without limitation, adrenocorticotropic hormone (ACTH), antidiuretichormone (vasopressin), atrial natriuretic peptide (ANP),cholecystokinin, follicle stimulating hormone (FSH), gastrin, glucagon,growth hormone, insulin, leptin, leuteinizing hormone (LH), oxytocin,prolactin, somatostatin, and thyroid stimulating hormone (TSH). Enzymesinclude, without limitation, acid alpha-glucosidase, adenosinedeaminase, alpha-galactosidase, alpha-L-iduronidase, arylsulfatase B,beta-galactosidase, beta-glucuronidase, galactose-6-sulfate sulfatase,glucocerebrosidase, heparan sulfamidase, heparan-alpha-glucosaminideN-acetyltransferase, hyaluronidase, iduronate-2-sulfatase,N-acetylgalactosamine-4-sulfatase, N-acetylglucosamine 6-sulfatase, andN-acetylglucosaminidase.

In some embodiments, the target polypeptide is a secreted protein.

In certain embodiments, the host cells are mammalian cells. In certainembodiments, the host cells are selected from the group consisting ofCHO cells, BHK-21 cells, NIH/3T3 cells, HEK293 cells, HeLa cells, SP2/0cells, NS0 cells, C127 cells, COS cells, Vero cells, and U937 cells. Allof these cells (cell lines) are commercially available from sources suchas American Type Culture Collection (ATCC, Manassas, VA). In certainembodiments, the host cells are selected from the group consisting ofCHO cells, HEK293 cells, and HeLa cells.

An aspect of the invention is a clonal population of transfected hostcells that express a selectable polypeptide and a target polypeptideobtainable by the method the invention. In certain embodiments, theclonal population of transfected host cells expresses a FACS-selectablepolypeptide and a target polypeptide obtainable by the method theinvention.

In certain embodiments, the clonal population yields a 2- to 30-foldimprovement in production of the target polypeptide compared to that ofa stable pool of transfected but uncloned host cells obtained at step(c).

For example, in some embodiments, the clonal population yields a 2- to30-fold, 3- to 30-fold, 5- to 30-fold, 10- to 30-fold, 15- to 30-fold,20- to 30-fold, 25- to 30-fold, 2- to 25-fold, 3- to 25-fold, 5- to25-fold, 10- to 25-fold, 15- to 25-fold, 20- to 25-fold, 2- to 20-fold,3- to 20-fold, 5- to 20-fold, 10- to 20-fold, 15- to 20-fold, 2- to15-fold, 3- to 15-fold, 5- to 15-fold, 10- to 15-fold, 2- to 10-fold, 3-to 10-fold, 5- to 10-fold, 2- to 5-fold, 3- to 5-fold, or 2- to 3-foldimprovement in production of the target polypeptide compared to that ofa stable pool of transfected but uncloned host cells obtained at step(c). In certain embodiments, the clonal population yields a greater than30-fold improvement in production of the target polypeptide compared tothat of a stable pool of transfected but uncloned host cells obtained atstep (c). For example, in certain embodiments, the clonal populationyields an up to 40-fold, up to 50-fold, up to 60-fold, up to 70-fold, upto 80-fold, up to 90-fold, or up to 100-fold improvement in productionof the target polypeptide compared to a stable pool of transfected butuncloned host cells obtained at step (c).

III. Producer Cells and Methods of Production Thereof

In some embodiments a heterogeneous population of producer cells isprovided. The heterogeneous population of producer cells can be producedusing any method known in the art or described herein. In someembodiments of any one of the methods provided, the heterogeneouspopulation of producer cells is produced by transfecting cells with avector that encodes the multicistronic mRNA and subjecting thetransfected cells to less than or equal to one round of medium-basedselection to select cells expressing varying levels (e.g., a variationof at least 10-, 100-, 1,000-, or 10,000-fold) of the multicistronicmRNA. In some embodiments, the vector further contains a drug-selectablemarker, e.g., a dihydrofolate reductase (DHFR) gene, and themedium-based selection is methotrexate (MTX, e.g., 1 nM-100 nM MTX),nucleotide-deficient medium, or a combination thereof. In someembodiments, the vector further contains a glutamine synthetase (GS)gene and the medium-based selection is methionine sulphoximine (MSX,e.g., 25-100 μM MSX). In some embodiments, the vector lacks adrug-selectable marker, e.g., lacks a DHFR gene or GS gene.

In some embodiments, FACS is used to select cells expressing varyinglevels of the multicistronic mRNA, e.g., by using the FACS selectablepolypeptide level to select the cells.

It will be readily apparent to those skilled in the art that othersuitable modifications and adaptations of the methods described hereinmay be made using suitable equivalents without departing from the scopeof the embodiments disclosed herein. Having now described certainembodiments in detail, the same will be more clearly understood byreference to the following examples, which are included for purposes ofillustration only and are not intended to be limiting.

EXAMPLES

The present invention is further illustrated by the following exampleswhich should not be construed as further limiting.

Example 1: Sorting for Early Post-Transfection Isolation of Cells(EPIC)—Proof of Concept

This example demonstrates the feasibility of a method of sorting totarget an unselected transfected early-expressing population for bulkenrichment prior to selection. This method of sorting is called “shortsorting” or “EPIC” (Early Post-transfection Isolation of Cells) and isdesigned to sort-isolate, or bulk enrich, early reporter expressionshortly after transfection. EPIC may significantly reduce selectiontimelines and/or improve productivity of the resulting heterogeneouspopulation. Experiments have been performed to investigate the reporterexpression profile of a transfected population throughout the course ofa nucleotide-deficient selection process. FIG. 1A depicts a generalscheme for EPIC. FIG. 1B shows the early expression of the reporter geneduring the nucleotide-deficient selection process. These offsethistograms demonstrate that early expression (e.g. day 3-4) is positiveand sortable; making isolating a sub-population of transfected cells foran EPIC process feasible.

As shown in FIG. 1A, EPIC can be executed by transfecting a populationand allowing early expression to develop, which can be targeted forisolation using flow cytometry or other means of cell sorting. Thesesort-isolated early-expression sub-populations can then be placed in aselection media to establish a stable expression pool. Isolation ofthese post-transfection early expression sub-populations prior toselection yields improved productivity over standardtransfection/selection methodologies alone (e.g., as shown in FIG. 1A).

To demonstrate proof of concept that detected CD52 signal is in factearly CD52 reporter expression, vectors directing expression of both redfluorescent protein (RFP) and CD52 (pGZ729-RFP) or RFP alone(pGZ700-RFP) were constructed and transfected for early expressionevaluation. In this system CD52 corresponds to the detectablepolypeptide, and RFP corresponds to the target polypeptide.

The pGZ729 vector backbone sequence (including sequence encoding CD52but not RFP) is shown below, followed by annotations of the sequence.

Sequence of pGZ729 expression vector (SEQ ID NO: 8):ggatccgctgtggaatgtgtgtcagttagggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccatagtcccgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgaggccgcctcggcctctgagctattccagaagtagtgaggaggcttttttggaggcctaggcttttgcaaaaagcttggggggggggacagctcagggctgcgatttcgcgccaaacttgacggcaatcctagcgtgaaggctggtaggattttatccccgctgccatcatggttcgaccattgaactgcatcgtcgccgtgtcccaaaatatggggattggcaagaacggagacctaccctggcctccgctcaggaacgagttcaagtacttccaaagaatgaccacaacctcttcagtggaaggtaaacagaatctggtgattatgggtaggaaaacctggttctccattcctgagaagaatcgacctttaaaggacagaattaatatagttctcagtagagaactcaaagaaccaccacgaggagctcattttcttgccaaaagtttggatgatgccttaagacttattgaacaaccggaattggcaagtaaagtagacatggtttggatagtcggaggcagttctgtttaccaggaagccatgaatcaaccaggccacctcagactctttgtgacaaggatcatgcaggaatttgaaagtgacacgtttttcccagaaattgatttggggaaatataaacttctcccagaatacccaggcgtcctctctgaggtccaggaggaaaaaggcatcaagtataagtttgaagtctacgagaagaaagactaacaggaagatgctttcaagttctctgctcccctcctaaagctatgcatttttataagaccatgggacttttgctggctttagatctttgtgaaggaaccttacttctgtggtgtgacataattggacaaactacctacagagatttaaagctctaaggtaaatataaaatttttaagtgtataatgtgttaaactactgattctaattgtttgtgtattttagattccaacctatggaactgatgaatgggagcagtggtggaatgcctttaatgaggaaaacctgttttgctcagaagaaatgccatctagtgatgatgaggctactgctgactctcaacattctactcctccaaaaaagaagagaaaggtagaagaccccaaggactttccttcagaattgctaagttttttgagtcatgctgtgtttagtaatagaactcttgcttgctttgctatttacaccacaaaggaaaaagctgcactgctatacaagaaaattatggaaaaatattctgtaacctttataagtaggcataacagttataatcataacatactgttttttcttactccacacaggcatagagtgtctgctattaataactatgctcaaaaattgtgtacctttagctttttaatttgtaaaggggttaataaggaatatttgatgtatagtgccttgactagagatcataatcagccataccacatttgtagaggttttacttgctttaaaaaacctcccacacctccccctgaacctgaaacataaaatgaatgcaattgttgttgttaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatottatcatgtctggatcctctacgccggacgcatcgtggccggcatcaccggcgccacaggtgcggttgctggcgcctatatcgccgacatcaccgatggggaagatcgggctcgccacttcgggctcatgagcgcttgtttcggcgtgggtatggtggcaggccgtggccgggggactgttgggcgccatctccttgcatgcaccattccttgcggcggcggtgctcaacggcctcaacctactactgggctgcttcctaatgcaggagtcgcataagggagagcgtcgaccgatgcccttgagagccttcaacccagtcagctccttccggtgggcgcggggcatgactatcgtcgccgcacttatgactgtcttctttatcatgcaactcgtaggacaggtgccggcagcgctctgggtcattttcggcgaggaccgctttcgctggagcgcgacgatgatcggcctgtcgcttgcggtattcggaatcttgcacgccctcgctcaagccttcgtcactggtcccgccaccaaacgtttcggcgagaagcaggccattatcgccggcatggcggccgacgcgctgggctacgtcttgctggcgttcgcgacgcgaggctggatggccttccccattatgattcttctcgcttccggcggcatcgggatgcccgcgttgcaggccatgctgtccaggcaggtagatgacgaccatcagggacagcttcaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctgcaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaacacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggccctttcgtcttcaagaattggggaccaagacagaaccataagccagtgggatagatcagaaatgttccagaggtgggatggggccagagtgcctgccccttgaaccgtcccagggaccagaggtgacaaagtggcaacacaggtcctgcctgggaatctggtctgctcctacttagtaaagctgcctggtgtcacacaagaggcccccacttattcctgcacccctggtggtaggtggcgtcttctcccctgcagccaccaggctcccctgagaacactgccggcagtcctcattgacaggcagtattcgctctgccccacccccacctgtgaattgcagggctggcaggtcctcaggcagctggcaaaccgcctgaacaactgagagatacagggccagggccagggcagtcccgtcccccggaggcagggaggggacgtgctgggaaagttctctctctcaggcccaggttggtgactgcagaaggcttctgtcaaatctcttttgtgggaaccacagagtagccctgaacgtgggggtgtgcttccagtatactctggggtcaccctttccatactggaggcctctgcaacttcaaaatgctctgctaccaacctagcacaaggaagttggtccagcctccccacgcagggccactgctgcagtccatatatggactaagccttccttggtttcaacacctacactcactgagcccctactatgtgtatgcagagccgagacaggccctgagcatctcatctgaagcacccttcttgcctaaattcagttttctgtcactttctcccaggaggtgtgtgtccctctaagctaagccaggggtccctcacccctgccccactcccatccctagtgtaggtatcagctgaagagcttcctgagcagaacactcttgggtgctgacattttgataaataggcccatgtttaggagagcaggggtccgggggcgggagatcttctctggtggattgagggctccaagaactactctttgagcacgctgcccctcccagagtccccacagcctccagatggactagaacacagttcggctgtggctgcacataactaacagaggatagatggtgggtcccagcccaacagtgcctggcaatcacccagagccaccagctaacggccttggcttagttttttgcctgggtgtgatcaggcagccctccaaaactgcccggactccatgacaagttttgcttgttctatagagcacagttcctttctaggtctggggcaagggacatcgggagacatcttcctgcaacagctccagtcactggaccaccaggctcgccctgtctttggtgtgtggccctgagtctcctaagtggcccaaacctgtgaagacccctccaaccacagttttgcttctaaattgtaccccaacacacctagcaaattgaaaccccaccagaagtcccccagatctggctttccggctattgctggcaagggggagtgactcccggcccattcaatccaggccccgcgtgttcctcaaacaagaagccacgtaaacataaaccgagcctccatgctgacccttgcccatcgaggtactcaatgttcacgtgatatccacacccagagggtcctggggtggggtcatgagccccagaatgcaggcttgataaccgagaccctgaatcgggcagtgtccacaagggcggaggccagtcatgcatgttcgggcctatggggccagcacccaacgccaaaactctccatcctcttcctcaatctcgctttctctctctctctctttttttttttttattttttttttttgcaaaaggaggggagagggggtaaaaaaatgctgcactgtgcggctaggccggtgagtgagcggcgcggagccaatcagcgctcgccgttccgaaagttgccttttatggctcgagtggccgctgtggcgtcctataaaacccggcggcgcaacgcgcagccactgtcgagtccgcgtccacccgcgagcacaggcctttcgcagctctttcttcgccgctccacacccgccaccaggtaagcagggacaacaggcccagccggccacagccctcccgtgggcagtgaccgcgctgcagggtcgcgggggacactcggcgcggacaccggggaaggctggagggtggtgccgggccgcggagcggacactttcagatccaactttcagtccagggtgtagaccctttacagccgcattgccacggtgtagacaccggtggacccgctctggctcagagcacgcggcttgggggaacccattagggtcgcagtgtgggcgctatgagagccgatgcagctttcgggtgttgaaccgtatctgcccaccttggggggaggacacaaggtcgggagccaaacgccacgatcatgccttggtggcccatgggtctttgtctaaaccggtttgcccatttggcttgccgggcgggcgggcgcggcgggcccggctcggccgggtgggggctgggttgccactgcgcttgcgcgctctatggctgggtattggggcgcgtgcacgctggggagggagcccttcctcttccccctctcccaagttaaacttgcgcgtgcgtattgagacttggagcgcggccaccggggttgggcgagggcggggccgttgtccggaaggggcggggtcgcagcggcttcggggcgcctgctcgcgcttcctgctgggtgtggtcgcctcccgcgcgcgcactagccgcccgccggcggggcgaaggcggggcttgcgcccgtttggggagggggcggaggcctggcttcctgccgtggggccgcctccggaccagcgtttgcctcttatggtaataacgcggccggcctgggcttcctttgtcccctgagtttgggcgcgcgccccctggcggcccgaggccgcggcttgccggaagtgggcagggcggcagcggctgcgcctagtggcccgctagtgaccgcgaccctcttttgtgccctgatatagttcgccggatcctaccgcggtagcggccgcgccaccttggagcgcttcctcttcctcctactcaccatcagcctcctcgttttggtacaaatacaaaccggactctccggacaaaacgacaccagccaaaccagcagcccctcagcatccagcaacataagcggaggcattttccttttcttcgtcgccaacgccataatccacctcttctgcttcagttgaaggccggccaatacgtaggcgcgccattgagtgagtgatttggcgcgccaagatatcacacccgggattaattaaaggtacctacgcgtagaattccacgtagtggtttaaactctagatactcgagggatctggatcataatcagccataccacatttgtagaggttttacttgctttaaaaaacctcccacacctccccctgaacctgaaacataaaatgaatgcaattgttgttgttaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatottatcatgtct

Elements of pGZ729 expression vector (and nucleotide locations):

Nucleotides 1-325—SV40 early promoter (for DHFR transcription)Nucleotides 347-1089—Dihydrofolate reductase (DHFR) open reading frameNucleotides 1090-1934—SV40 early intron and polyANucleotides 2684-3366—E. coli ColE1 originNucleotides 3464-4123—Ampicillin resistance geneNucleotides 4528-7539—Hamster (3-actin promoter (for transcription ofgene of interest)Nucleotides 7568-7753—CD52 open reading frame (containing TTG startcodon)Nucleotides 7781-7791—Stop codons in each of 3 reading framesNucleotides 7813-7872—Multiple cloning site (for insertion of targetpolypeptide with ATG start codon)Nucleotides 7882-8123—SV40 early polyA

As shown in FIG. 2 , CHO cells transfected with pGZ729-RFP producedearly expression of both CD52 and RFP that peaked around days 2 and 3,with signal deteriorating out to day 7 post-transfection. Therefore,EPIC targeting on or near days 2-3 is suitable for isolation ofearly-expressing sub-populations of transfected host cells. In order todemonstrate that these relatively low fluorescence intensity signalswere in fact CD52 expression, CHO cells transfected with eitherpGZ729-RFP or pGZ700-RFP were analyzed for RFP and CD52 expression. Asshown in FIG. 3 , both CHO cells transfected with pGZ729-RFP and CHOcells transfected with pGZ700-RFP robustly expressed RFP (top left andbottom left, respectively), whereas while CHO cells transfected withpGZ729-RFP had modest expression of CD52 (top right), CHO cellstransfected with pGZ700-RFP expressed essentially no detectable CD52(bottom right). These findings support the notion that these relativelylow fluorescence intensity signals were in fact CD52 expression from thealternate start expression cassette, and are suitable targets forsorting isolation (EPIC).

As a further proof of principle, early RFP expression was targeted forEPIC from a transfected population using pGZ729-RFP to then generate astable pool. Early RFP expression was targeted for sort isolation andcollection two days after transfection (peak transience). The EPICgenerated RFP positive sub-population was then used to establish astable pool via selection in 0 nM MTX nucleotide deficient media. Astandard transfection/selection pool was also generated to serve ascomparative control. As shown in FIG. 7 , the EPIC generated pool (whichtargeted early RFP expression) yielded a stable pool (EPIC pool) withgreater RFP and CD52 reporter expression than traditionaltransfection/selection methodology alone (0 nM pool). Resultsdemonstrate a FLARE independent proof of principle supporting the claimthat EPIC generated pools are more productive than traditionaltransfection/selection methodologies.

Example 2: Sorting for Early Post-Transfection Isolation of Cells(EPIC)—Producer Cell Pool Generation

EPIC was initially attempted using mAb #1 in which CHO cells weretransfected and given 2 days to recover, after which 0 nM MTX selectionwas initiated to establish early expression. Four days aftertransfection, early expression of CD52 cell surface reporter wastargeted for sort isolation (EPIC). Sorting targeted only positiveexpression which was collected as a bulk enriched population of about 1million cells which was then allowed to continue selection innucleotide-deficient media (0 nM MTX). As a control, a non-sortedtransfection was allowed to continue selection via standard selectionprocedure. As shown in FIG. 4 , by day 8 sorting for EPIC yielded aslightly enriched population as seen by CD52 reporter expression ascompared to standard selection. As selection of both populationscontinued, however, this small EPIC sub-population became more prominentover time. In fact, the CD52-negative sub-population was all buteliminated upon selection completion. Comparatively, the standardselection method demonstrated a slight improvement in CD52 reporterexpression which is historically typical. Sorting for EPIC, or isolatingearly expression, yielded a sub-population of positive expression thathad a preferential survivability over the less expressive cells, whichin turn yielded a more productive stable pool.

The EPIC and standard selected pools were both used to establish unfedbatch cultures to determine mAb #1 titers. As shown in FIGS. 4 and 5 ,the EPIC-generated pool yielded a titer of 502 mg/L, far outpacing anypools generated by MTX amplification, again using no MTX throughout theprocesses. Comparatively, the pool generated by standard selectionyielded a titer of 150 mg/L, which was 3-fold lower than that of theEPIC-generated pool.

While these initial sorts targeting EPIC took 35 days(transfection/sort/isolation) to achieve completion to a stable pool,this was directly related to the small number of sorted cells collected(1 million) which then had to endure both the expansion and selection toa stable population. Such timelines could be greatly reduced by eithersimply collecting more cells and/or targeting a purer sort. Many of thecells sorted had high levels of impurities (cells with little to noexpression) and had to be selected (killed) out, prolonging theselection/expansion times.

Example 3: Sorting for Early Post-Transfection Isolation of Cells(EPIC)—Clone Generation

The EPIC-generated pool of Example 2 was next used to generate clonesusing FLARE as previously described (see, e.g., Cairns, V. et al. (2011)Utilization of Non-AUG Initiation Codons in a Flow Cytometric Method forEfficient Selection of Recombinant Cell Lines, Biotechnol Bioeng108(11):2611-2622). Briefly, FLARE was used to isolate and single cellplate the top 3-5% of reporter-expressing cells from each pool usingFACS. Expanded clones were then screened (taking top 30% positiveexpressers), again using FLARE, to identify only the top tier clones toexpand for target polypeptide titer evaluation. As shown in FIG. 6 , topexpressing EPIC-generated clones achieved similar titers to those ofbest clones from traditional methods, e.g., using MTX-amplified pools(near 2.0 g/L). Results demonstrated that using EPIC to isolate earlyexpression populations prior to selection is a viable alternative totraditional transfection and selection methodologies. EPIC offers aMTX-independent methodology to achieve clone titers similar to thosefrom traditional MTX methodologies, resulting in potentially more robustand stable clones. Alternatively, EPIC is also amenable to MTXintroduction during selection/expansion of EPIC-generatedsub-populations, with the potential to drive even higher expression inthese enriched populations.

Example 4: Early Post-Transfection Isolation of Cells (EPIC) for CloneGeneration—Head-to-Head Comparison to Other Methods

A comprehensive study was performed to directly compare the EPIC process(MTX-independent), the Rapid Bulking process (MTX-independent), and astandard MTX selection process (Direct Selection process) for clonegeneration. Three independent experiments were performed for eachprocess, each experiment using a different recombinant protein (twomonoclonal antibodies and one Fc fusion protein). FIG. 8 depicts thegeneral scheme for the comparative study, with each of the threeprocesses being initiated from a single source pool of transfectedcells.

In the “EPIC Process,” one portion of the pooled transfected cells wasallowed to continue recovery for two additional days prior to sortisolation of the cell surface reporter positive expression population.The sort isolated cells were placed in nucleotide deficient media (noMTX) for expansion to generate a stable EPIC pool. In the “Rapid BulkingProcess,” one portion of the pooled transfected cells was placed innucleotide deficient media (no MTX) to generate a population which wasthen bulk enriched two times (targeting top 10%), by sort isolationbased on cell surface reporter expression levels ending with the rapidbulk #2 pool. In the “Direct Selection Process,” one portion of thepooled transfected cells was placed in media with MTX for a standardselection procedure. The resulting pool was passaged in nucleotidedeficient media prior to clone generation. Each arm represents anindependent method to prepare a stably transfected population of cells(pools) suitable for clone generation.

For each of the three independent processes, the pool generated at theend of the process was used to generate clones using FLARE as previouslydescribed (see, e.g., Cairns, V. et al. (2011) Utilization of Non-AUGInitiation Codons in a Flow Cytometric Method for Efficient Selection ofRecombinant Cell Lines, Biotechnol Bioeng 108(11):2611-2622). Briefly,FLARE was used to isolate and single cell plate the top 3-5% ofreporter-expressing cells from each pool using FACS. Expanded cloneswere then screened (taking the top 30% of positive expressers), againusing FLARE, to identify only the top tier clones to expand for targetpolypeptide titer evaluation.

FIG. 9 shows the distribution of target polypeptide (mAb #1, mAb #2, orFc Fusion #1) titers for the clones generated using the threeindependent processes.

The results for mAb #1 and mAb #2 clearly demonstrate that clonesgenerated from EPIC and rapid bulking processes, both of which do notuse MTX, have similar productivity to clones generated from a standardMTX selection process. For the Fc fusion protein, the range of clonetiters from the EPIC process is similar to that from the rapid bulking(no MTX) and standard MTX selection processes except at the top end ofthe range, the best EPIC clone titers are lower than the best clonetiters from the other two processes. Despite this, the EPIC processstill generated clones with titers up to 1.8 g/L (in uncontrolled batchculture). Also, Fc fusion protein clones from the rapid bulking process(no MTX) achieved titers as high as those from the MTX selectionprocess. While there is a greater frequency of higher producersassociated with the MTX selection process, this may indicate that theMTX process results in a less diverse group of clones, perhaps being ofsimilar genetic origin with similar transgene integrations.Alternatively, the EPIC and rapid bulking methods, which are sortenrichment processes rather than a drug selection/cell death process,may yield a more diverse group of clones with different transgeneintegration sites and therefore a greater range of productivity. Agreater degree of clone diversity is often preferable when identifyingcell lines that are suitable for a manufacturing process.

This example also demonstrates the significant time savings achieved bythe EPIC process. FIG. 10 shows the number of days from transfection tothe pool used for cloning (black) and from the pool to the final clones(gray).

Regarding the time from transfection to final pool, the EPIC processgenerates the pools one month faster than the standard MTX selectionprocess. It is at this pool generation step that the most significanttime savings is realized, and the faster generation of pools with thisMTX-independent methodology translates to an overall CLD processtimeline reduced by 1 month.

This example demonstrates that the MTX-independent EPIC methodology cangenerate clones with comparable productivity and significantly reducedtimelines compared to a standard MTX selection process. In addition tobeing a more rapid process, the absence of MTX could result in morerobust and stable clones compared to similarly productive clones from aMTX process.

Example 5: Variable Sort Targeting for Early Post-Transfection Isolationof Cells (EPIC)—EPIC is Isolating Transiently Positive Subpopulations

Experiments were designed to gain a better understanding of how EPICworks. It was hypothesized that sorting cells early after transfectionis targeting and isolating a subpopulation(s) of highly positivetransient expression. However, it could not be ruled out that targetingreporter expression in this time frame could also be isolating cellswith early stable integration (in the cellular genome) which would helpyield significantly enriched EPIC pools. Two experiments were performedto demonstrate that EPIC results from isolation of transient expressionsubpopulations. First, sorting for EPIC was performed over a time courseto show that isolations during the period of peak transient expression(days 2 to 4; see FIG. 2 ) yielded optimal EPIC pool enrichments.

Secondly, variable sort targeting for EPIC was performed at 2 dayspost-transfection, with the aim of isolating an increasingly positivetransient expression population to, in turn, yield a further enrichedEPIC pool.

Using a day 2 (peak) transient expression population, independent sortstargeted a full positive transient expression population (+) and a morehighly positive transient expression subpopulation (++). The dataconclusively demonstrate that isolation of a more positive transientsubpopulation results in greater enrichment in the final EPIC pool thatwas generated. Being at day 2 post-transfection, there would not yet becells with stably integrated expression as this is prior to the loss oftransient expression and appearance of stable expression (FIG. 2 ).Therefore, the correlative relationship (FIGS. 11A and 11B) at thisspecific early time point provides evidence that EPIC pool enrichmentsresult directly from transient subpopulation isolations from peak (ornear peak) transient expression shortly after transfection.

1. A method of producing a population of producer cells expressing atarget polypeptide, the method comprising: (a) transfecting host cellswith one or more vectors that encode one or more mRNAs, wherein the oneor more mRNAs encode a selectable polypeptide and the targetpolypeptide; (b) isolating from the transfected host cells, within 2 to15 days of transfection, a sub-population of early-expressingtransfected host cells which express the selectable polypeptide; and (c)expanding the sub-population of transfected host cells, therebyproducing a population of producer cells expressing the targetpolypeptide.
 2. The method of claim 1, wherein steps (b) and (c) areeach performed in drug-selection-free medium.
 3. The method of claim 1,further comprising isolating the target polypeptide from the populationof producer cells.
 4. The method of claim 1, further comprisingisolating one or more single transfected host cells from the expandedsub-population and culturing the one or more single transfected hostcells to produce clonal populations of the one or more singletransfected host cells.
 5. The method of claim 4, wherein at least oneof the clonal populations of the one or more single transfected hostcells yields a 2- to 30-fold improvement in production of the targetpolypeptide compared to that of a stable pool of transfected butuncloned host cells obtained at step (c).
 6. (canceled)
 7. (canceled) 8.The method of claim 1, wherein the isolating in step (b) is performedbetween two and four days, two days, or three days after transfection.9-11. (canceled)
 12. The method of claim 1, wherein the expanding instep (c) is for between 4-31 days.
 13. (canceled)
 14. (canceled)
 15. Themethod of claim 1, wherein the isolating in step (b) employs magneticactivated cell sorting (MACS), fluorescence activated cell sorting(FACS), or ClonePix.
 16. The method of claim 1, wherein the selectablepolypeptide is a FACS=selectable polypeptide and the isolating in step(b) employs FACS.
 17. The method of claim 1, wherein the targetpolypeptide and the selectable polypeptide form a fusion polypeptide.18. The method of claim 1, wherein the target polypeptide and theselectable polypeptide are encoded by a single multicistronic mRNA. 19.The method of claim 18, wherein the multicistronic mRNA comprises afirst open reading frame (ORF) that encodes the selectable polypeptideand a second ORF that encodes the target polypeptide, wherein the firstORF is 5′ to the second ORF.
 20. The method of claim 19, wherein thefirst ORF has a non-AUG start codon.
 21. The method of claim 20, whereinthe non-AUG start codon is a UUG, GUG, or CUG in a Kozak consensussequence.
 22. The method of claim 19, wherein the second ORF has an AUGstart codon.
 23. The method of claim 19, wherein the ORF that encodesthe selectable polypeptide is devoid of any AUG sequences.
 24. Themethod of claim 1, wherein the selectable polypeptide is CD52 or CD59.25. The method of claim 1, wherein the target polypeptide is atherapeutic agent, a secreted protein or an antibody or an Fc fusionprotein.
 26. (canceled)
 27. (canceled)
 28. The method of claim 1,wherein the host cells are selected from the group consisting of CHOcells, HEK293 cells, and HeLa cells.
 29. A clonal population oftransfected host cells that express a selectable polypeptide and atarget polypeptide obtainable by the method of claim
 1. 30. (canceled)